An ever-expanding field of applications exists for rapid, highly specific, sensitive, and accurate methods of detecting and quantifying chemical, biochemical, and biological substances, including enzymes such as may be found in biological samples. Because the amount of a particular analyte of interest such as an enzyme in a typical biological sample is often quite small, analytical biochemists are engaged in ongoing efforts to improve assay performance characteristics such as sensitivity.
One approach to improving assay sensitivity has involved amplifying the signal produced by a detectable label associated with the analyte of interest. In this regard, luminescent labels are of interest. Such labels are known which can be made to luminesce through photoluminescent, chemiluminescent, or electrochemiluminescent techniques. "Photoluminescence" is the process whereby a material luminesces subsequent to the absorption by that material of light (alternatively termed electromagnetic radiation or emr). Fluorescence and phosphorescence are two different types of photoluminescence. "Chemiluminescent" processes entail the creation of the luminescent species by a chemical reaction. "Electrochemiluminescence" is the process whereby a species luminesces upon the exposure of that species to electrochemical energy in an appropriate surrounding chemical environment.
The signal in each of these three luminescent techniques is capable of very effective amplification (i.e., high gain) through the use of known instruments (e.g., a photomultiplier tube or pmt) which can respond on an individual photon by photon basis. However, the manner in which the luminescent species is generated differs greatly among and between photoluminescent, chemiluminescent, and electrochemiluminescent processes. Moreover, these mechanistic differences account for the substantial advantages as an bioanalytical tool that electrochemiluminescence [hereinafter, sometimes "ECL"] enjoys vis a vis photoluminescence and chemiluminescence. Some of the advantages possible with electrochemiluminescence include: (1) simpler, less expensive instrumentation; (2) stable, nonhazardous labels; and (3) increased assay performance characteristics such as lower detection limits, higher signal to noise ratios, and lower background levels.
As stated above, in the context of bioanalytical chemistry measurement techniques, electrochemiluminescence enjoys significant advantages over both photoluminescence and chemiluminescence. Moreover, certain applications of ECL have been developed and reported in the literature. U.S. Pat. Nos. 5,147, 806; 5,068,808; 5,061,445; 5,296,191; 5,247,243; 5,221,605; 5,238,808, and 5,310,687, the disclosures of which are incorporated by reference, detail certain methods, apparatuses, chemical moieties, inventions, and associated advantages of ECL.
Copending and commonly-assigned U.S. patent application Ser. No. 08/368,429, filed January 4, 1995, the disclosure of which is incorporated by reference, details certain aspects of ECL in connection with beta-lactam and beta-lactamase (neither of which is conjugated through a covalent linkage to an electrochemiluminescent compound).
None of the above-identified documents disclose nor suggest the present invention. Additionally, the practice of the invention offers significant advantages to the skilled bioanalytical chemist in comparison to the electrochemiluminescent techniques taught by these documents. Accordingly, the invention meets the as-yet unmet needs of skilled workers with respect to the achievement of improved assay performance characteristics (e.g., signal output, detection limits, sensitivity, etc.) for the measured species and represents a patentable advance in the field.
Assays based on ECL are well known in the art and are finding expanding applications because of their accuracy, ease of use and freedom from radioactive materials.
A particularly useful ECL system is described in a paper by Yang et al, Bio/Technology, 12, pp. 193-194 (Feb. 1994). See also a paper by Massey, Biomedical Products, October 1992 as well as U.S. Pat. Nos. 5,235,808 and 5,310,687, the contents of these papers and patents being incorporated herein by reference.
ECL processes have been demonstrated for many different molecules by several different mechanisms. In Blackburn et al (1991) Clin. Chem. 37/9, pp. 1534-1539, the authors used the ECL reaction of ruthenium (II) tris(bipyridyl), Ru(bpy).sub.3.sup.+2, with tripropylamine (TPA) (Leland et al (1990) J. Electrochem. Soc. 137:3127-31) to demonstrate the technique. Salts of Ru(bpy).sub.3.sup.+2 are very stable, water-soluble compounds that can be chemically modified with reactive groups on one of the bipyridyl ligands to form activated species with which proteins, haptens, and nucleic acids are readily labeled. The activated form of the Ru(bpy).sub.3.sup.+2 used by Blackburn et al was Ru(bpy).sub.3.sup.+2 -NHS ester:
Beta-lactamases which hydrolyze the amide bonds of the .beta.-lactam ring of sensitive penicillins and cephalosporins are widely distributed amongst microorganisms and play a role in microbial resistance to P-lactam antibiotics. Beta-lactamases constitute a group of related enzymes which are elaborated by a large number of bacterial species but not by mammalian tissues and can vary in substrate specificities. See generally Payne, D.J., J. Med. Micro (1993) 39, pp. 93-99: Coulton, S. & Francois, L., Prog. Med. Chem. (1994) 31, 297-349; Moellering, R.C., Jr., J. Antimicrob. Chemother. (1993) 31 (Suppl. A), pp. 1-8: and Neu, H.C., Science (1992) 257, pp. 1064-1072.
The detection of .beta.-lactamase activity in a body fluid has long been considered to be indicative of a recent or current bacterial infection.
The developing microbial resistance to antibiotics such as penicillin and cephalosporin has been of concern for awhile. Recently, this concern has escalated in light of the dwindling number of new antibiotics and the over-use of those which are known. It is becoming more imperative to select the optimum antibiotic for treating a particular infection and to avoid prescribing the latest antibiotic when effective alternatives exist. This ability to select the optimum antibiotic is especially critical in those facilities involved in long-term care facilities where antibiotic resistance is increasingly becoming a problem. The lifetime of the current family of antibiotics can be prolonged by the selection of the optimum antibiotic. See generally Harold C. Neu, "The Crisis in Antibiotic Resistance", Science Vol. 257 (Aug. 11, 1992) pp. 1064-1072.
The rising resistance to microbial resistance to antibiotics has heightened the need for a test which can rapidly measure quantitatively the degree of resistance to a particular .beta.-lactam antibiotic such as a penicillin or a cephalosporin and then select the most appropriate antibiotic for a particular infective condition.
Several methods currently exist for the detection of microbial .beta.-lactamases. Some representative examples follow.
W.L. Baker, "Co-existence of .beta.-lactamase and penicillin acylase in bacteria: detection and quantitative determination of enzyme activities", J. Appl. Bacteriol. (1992) Vol. 73, No. 1, pp. 14-22 discloses a copper-reducing assay for the detection of penicilloates and a fluorescamine assay to detect 6-aminopenicillanic acid concentrations when both substances were produced by the action of the enzymes on a single substrate.
U.S. Pat. No. 5,264,346 discloses a calorimetric assay for .beta.-lactamase which has a variety of applications. The assay is based on the decolorization of a chromophore formed by oxidation of either the N-alkyl derivative of p-phenylenediamine or the 3,3',5,5'-tetraalkyl derivative of benzidine. The decolorization is attributed to the presence of an open .beta.-lactam ring product resulting from the hydrolysis of cephalosporin or penicillin. Decolorization with the open .beta.-lactam product of penicillin requires the presence of a decolorization enhancer such as mercury containing compounds. The enhancer is not required for decolorization with the open .beta.-lactam product of cephalosporin.
U.S. Pat. No. 4,470,459 discloses a rapid method for the detection of the presence of .beta.-lactamase from microbial sources which is based on a .beta.-lactamase conversion of a .beta.-lactam substrate which reverses its ability to fluoresce. Specific .beta.-lactams mentioned as having this property include ampicillin, cephalexin, amoxicillin, cefadroxil and cephaloglycin. The change in the ability to fluoresce is attributed to the presence of .beta.-lactamase.
WO 84/03303 discloses a microbiological test process for identifying producers of .beta.-lactamase. The assay relies on changes in acidity which affect the fluorescence of the indicator such as coumarin. This change in acidity is attributed to the conversion product produced by the presence of the .beta.-lactamase.
A.C. Peterson et al, "Evaluation of four qualitative methods for detection of .beta.-lactamase production in Staphylococcus and Micrococcus species", Eur. J. Clin. Microbiol. Infect. Dis. (1989), Vol. 8, No. 11, pp. 962-7 presents certain factors which were employed in evaluating qualitative assays for .beta.-lactamase.
Robert H. Yolken et al, "Rapid diagnosis of infections caused by .beta.-lactamase-producing bacteria by means of an enzyme radioisotopic assay", The Journal of Pediatrics, Vol. 97, No. 5 (Nov. 1980) pp. 715-720 discloses a sensitive enzymatic radioisotopic assay for the measurement of .beta.-lactamase as a rapid test for detection of bacterial infection. The assay protocol involves an incubation step with sample followed by the separation step on a positively charged column such as DEAE-Sephacel prior to measurement of the radioactivity of eluted fractions. The .beta.-lactamase converted penicillinic product has an additional carboxyl group which insures its stronger binding to the positively charged column than the penicillin. Differences in radioactivity between the eluted fractions and the original values are attributed to the presence of .beta.-lactamase.
Prior to the invention disclosed herein, there remains a need for a universal assay for .beta.-lactams and .beta.-lactamases which is both very rapid (10 minutes or less) and also very sensitive.
The invention disclosed within this application achieves these needs by adapting electrochemiluminescence methodologies to the measurement of .beta.-lactams or .beta.-lactamases. Other objects of the invention will also be apparent from the description of the invention which follows.